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Solar ultraviolet radiation (UV) can cause DNA damage in microorganisms. Flap endonuclease 1 (FEN1) is a structure-specific nuclease and plays important roles in DNA replication and repair. At present, the properties and functions of FEN1 have not been characterized in detail in invertebrates such as Bombyx mori. In this study, Bombyx mori FEN1 (BmFEN1) was expressed in E. coli, and was shown to have nuclease activity that nonspecifically cleaved DNA in vitro. However, inside the cell, BmFEN1 did not cleave DNA randomly. Truncated BmFEN1 missing the nuclear localization signal (346-380 aa) still had the nuclease activity, but was no longer precisely localized to the sites of UV-induced DNA damage. It was further found that BmFEN1 favored the faster repair of UV-damaged DNA. The present study will provide a reference for further understanding the functions of BmFEN1 and UV-induced DNA damage repair mechanisms in insects., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

At present, the effect of ultraviolet (UV) radiation on the interaction between Bombyx mori nucleopolyhedrovirus (BmNPV) and host remains unclear. In the current study, UV treatment significantly reduced the activity of BmNPV budded viruses (BVs), and UV-damaged BmN cells were not conducive to BmNPV proliferation. BmNPV infection significantly reduced the viability of host cells, but increased the viability of high-dose UV-treated host cells. Furthermore, the quantitative reverse-transcription PCR (qPCR) results suggested that BmNPV and Bombyx mori might mutually use the same DNA repair proteins for repairing UV-induced damage and BmNPV infection promote the ability of host cells to repair UV-induced damage., (© 2019 Wiley Periodicals, Inc.)

Orf101 (Bm101) of Bombyx mori nucleopolyhedrovirus (BmNPV) is a highly conserved gene in lepidopteran nucleopolyhedroviruses, but its function remains unknown. In this study, Bm101 was characterized. Transcripts of Bm101 were detected from 24 through 96 h post infection (h p.i.) by RT-PCR. The corresponding protein was also detected from 24 to 96 h p.i. in BmNPV-infected BmN cells by Western blot analysis using a polyclonal antibody against Bm101. Western blot assay of occlusion-derived virus and budded virus (BV) preparations revealed that Bm101 encodes a 28-kDa structural protein that is associated with BV and is located in the envelope fraction of budded virions. In addition, confocal analysis showed that the protein was localized in the cytosol and cytoplasmic membrane in virus-infected cells. In conclusion, the available data suggest that Bm101 is a functional ORF of BmNPV and encodes a protein expressed in the late stage of the infection cycle that is associated with the BV envelope.

To investigate whether Bombyx mori immunized with Bacillus subtilis spore displaying GP64 escape from the B. mori nucleopolyhedrovirus (BmNPV) attack, a recombinant integrative plasmid named pJS700-GP64 was constructed, which carries a recombinant cotC-Gp64 gene under the control of the cotC promoter. In this study, pJS700-GP64 was transformed into B. subtilis 168 (trp(-)) competent cells, an amylase (amyE) inactivated mutant was selected, and was confirmed to be a double cross-over integrant, cotC-Gp64 fragment of which was integrated into B. subtilis chromosome. Gp64 was expressed on the spore surface and recognized by Gp64-specific antibody. Results of B. mori when challenged with BmNPV indicated that B. mori vaccinated with the recombinant spores possessed resistance to the invasion of BmNPV at some degree.

Eukaryotic DNA polymerase δ (pol δ) plays a crucial role in chromosomal DNA replication and various DNA repair processes. It is thought to consist of p125, p66 (p68), p50 and p12 subunits. However, rigorous isolation of mammalian pol δ from natural sources has usually yielded two-subunit preparations containing only p125 and p50 polypeptides. While recombinant pol δ isolated from infected insect cells have some problems of consistency in the quality of the preparations, and the yields are much lower. To address these deficiencies, we have constructed recombinant BmNPV baculoviruses using MultiBac system. This method makes the generation of recombinant forms of pol δ containing mutations in any one of the subunits or combinations thereof extremely facile. From about 350 infected larvae, we obtained as much as 4 mg of pol δ four-subunit complex. Highly purified enzyme behaved like the one of native form by rigorous characterization and comparison of its activities on poly(dA)/oligo(dT) template-primer and singly primed M13 DNA, and its homogeneity on FPLC gel filtration. In vitro base excision repair (BER) assays showed that pol δ plays a significant role in uracil-intiated BER and is more likely to mediate LP BER, while the trimer lacking p12 is more likely to mediate SN BER. It seems likely that loss of p12 modulates the rate of SN BER and LP BER during the repair process. Thus, this work provides a simple, fast, reliable and economic way for the large-scale production of human DNA polymerase δ with a high activity and purity, setting up a new platform for our further research on the biochemical properties of pol δ, its regulation and the integration of its functions, and how alterations in pol δ function could contribute to the etiology of human cancer or other diseases that can result from loss of genomic stability.

Orf94 (Bm94) of Bombyx mori nucleopolyhedrovirus (BmNPV) potentially encodes 424-amino acids with a predicted molecular weight of 49.4 kDa, but its function remains unknown. Blast search results revealed that Bm94 homologues exist in 10 completely sequenced Lepidopteron NPVs with identities ranging from 95 to 37%. Results of our recent study showed that Bm94 was transcribed from 12 to 72 h and the corresponding protein was detected from 24 to 72 h post-infection. Furthermore, Western blot analysis revealed that Bm94 was present in occlusion-derived virus (ODV) and in total protein from BmNPV-infected BmN cells, but not in budded virus. Immunofluorescence analysis revealed that the protein located primarily in the cytoplasm and was also present in the nucleus in the later infection. In conclusion, these results together indicated that Bm94 was a late gene, which distributed both in the cytoplasm and in the nucleus, and was identified to be a component of BmNPV ODV.

Calorie restriction (CR) is known to extend life span from yeast to mammals. To gain an insight into the effects of CR on growth and development of the silkworm Bombyx mori at protein level, we employed comparative proteomic approach to investigate proteomic differences of hemolymph and fat body of the silkworm larvae subjected to CR. Thirty-nine differentially expressed proteins were identified by MALDI TOF/TOF MS. Among them, 19 were from the hemolymph and 20 from the fat body. The hemolymph of the CR group contained two down-regulated and 17 up-regulated proteins, whereas the fat body contained 15 down-regulated and five up-regulated ones. These proteins belonged to those functioning in immune system, in signal transduction and apoptosis, in regulation of growth and development, and in energy metabolism. Our results suggest that CR can alter the expression of proteins related to the above four aspects, implying that these proteins may regulate life span of the silkworm through CR.

NS1 gene of Bombyx mori parvo-like virus (China Zhenjiang isolate, BmDNV-Z) codes a predicted 316-amino acid protein, but its function remains unknown. Results of the current study showed that purified recombinant 6 x His-NS1 protein possesses ATP binding, ATPase, DNA binding, and helicase activities. Only one protein was captured in infected Bombyx mori midgut cells against NS1 target protein by employing co-immunoprecipitation, which was identified to be a viral protein by mass spectrometry. The NS1-interacting protein is encoded by BmDNV-Z ORF4 and its molecular is about 100 kD. Analysis of His pull-down confirmed that binding of identified viral protein to purified recombinant 6 x His-NS1 protein in vitro. Taken together, our results indicated that BmDNV-Z NS1 was a multifunctional protein, which may be involved with virus replication.

The vacuolar ATPase (V-ATPase) is a multifunctional enzyme that consists of several subunits. Subunit B is a part of the catalytic domain of the enzyme. The result of the RT-PCR suggested that the V-ATPase B subunit is a ubiquitous gene. 24 h after the larvae were infected with the Bombyx mori nucleopolyhedrovirus (BmNPV), the expression level of the V-ATPase B subunit in the midgut of the resistant strain NB was about 3 times higher than in the susceptible strain 306, and then the expression level of the V-ATPase B subunit decreased rapidly to a very low level. This indicated that the virus may cause a lot of changes of physiological conditions in the midgut. Localization of the V-ATPase B subunit was attempted in midgut cells of Bombyx mori by immunohistochemistry. The immunohistochemical localization with the antibody against the B subunit revealed a positive staining in goblet cell apical membranes of Bombyx mori midgut cells as well as in the midgut of Manduca sexta. This sequence has been registered in GenBank under the accession number EU727173.

Nomuraea rileyi (N. rileyi) is the causative agent of the silkworm, Bombyx mori, green muscardine which can cause severe worldwide economical loss in sericulture. Little is known about N. rileyi at the protein level for this entomopathogenic parasite which belongs to the Ascomycota. Here, we employed proteomic-based approach to identify proteins of N. rileyi spores collected from the dead silkworm. In all, 252 proteins were separated by two-dimensional gel electrophoresis (2-DE), and were subjected to mass spectrometry (MS) analysis, 121 proteins have good MS signal, and 24 of them were identified due to unavailability of genomic information from N. rileyi. This data will be helpful in understanding the biochemistry of N. rileyi.

The nanos gene encodes a zinc-finger protein which is required for the migration and differentiation of primordial germ cells as well as for their fate maintenance. In this study, a 1913 bp nanos gene was cloned and characterized in silkworm (Bombyx mori). RT-PCR and Western blot analysis showed that the nanos was expressed in developing embryos and various silkworm larval tissues. The expression patterns of Nanos and Vasa in silkworm larval gonads were analyzed using immunohistochemistry. It was found that, in silkworm larval ovaries, the Nanos and Vasa proteins were expressed in oocytes. While in testes, high expression of Nanos and Vasa was detected in spermatogonia and relatively weaker expression was found in spermatocytes at latter stages.